1. Field of the Invention
The invention relates to the destruction or inactivation of microbes by subjecting them to a high potential gradient.
2. Description of the Related Art
As shown by publications in the open technical literature (see Technical Bibliography, below) it has been known for at least fifty years that microbes can be destroyed or deactivated by high potential gradients. In the earliest publications (1949-1965) practical application of this phenomenon for the purpose of disinfecting liquids--and liquid foods in particular--was emphasized. Somewhat later (1970-1985), carefully controlled application of high potential gradients to the manipulation of biological cells was studied and reported. Publications described, inter alia, the use of high potential gradients to render the membranes of biological cells permeable, to organize a number of cells into a group and to accomplish the fusion of two or more cells. Practical devices to accomplish these things were developed and offered for sale as commercial products for use in biological laboratories. The microbiological studies associated with this work provided valuable fundamental information on the effect of high potential gradients on cells. Among other things, it became evident that high potential gradients produce porosity and, in some cases, permanent damage to cell membranes by exerting electromechanical force (electrophoretic force) on the membrane. In other words, the observed effects were due to these forces and not due to electric currents or ohmic heating.
While some microbes--such as spores, in particular,--can be found in gaseous media (in the air, for example) or in solid materials or on the surfaces of solid materials, the vital processes of most microbes require a liquid medium--usually an aqueous medium. Such liquid media are usually weak electrolytes (water being an example) and consequently these media have comparatively high electrical conductivity. Conductivity of 0.05 Siemens (S) per meter is a typical value; but values differing from this by a factor of 10.sup.6 would still be classified as "semi-conducting", or "non-insulating". Consequently, it was recognized from the earliest work (1949-1960) that high potential gradients could be applied successfully to media containing microbes only under certain special conditions. A potential gradient of 10.sup.6 volts/meter is of the order of the lowest gradient which will have a permanent effect on a microbe. Such a gradient, applied as a steady (DC) gradient to a medium having a conductivity of 0.05 S/m, would result in a current density of 5.times.10.sup.4 amp/m.sup.2, with consequent power dissipation of 50,000 Megawatts/m.sup.3 ; and the temperature of the medium would rise at an initial rate of roughly 12,000.degree. C./sec. Of course, these considerations were well-known to all who have worked on this subject. Some of the earliest work applied high-frequency AC voltages (Burton--1949; Doevenspeck--1961) but by 1965, pulsed voltages had become accepted as the preferred means of creating high potential gradients in the various media which were studied. (E.g., Hamilton & Sale--1967; Sale & Hamilton--1967,1968). Pulse lengths in the range 0.1 microsec to several milliseconds were employed. It was established that the effect of potential gradients on cells--as measured by the induced porosity of the membrane or by the lethal effect on the cells--increased rapidly as the magnitude of the gradient increased. Sale and Hamilton (1968) presented a formula for the potential difference across a microbe in an electric field which has been widely used ever since. They assumed a spherical cell of radius a.sub.0 in an electrolytic medium (specific resistance .rho. and dielectric constant .kappa.). The cell membrane is assumed to have a very high resistance--high enough that it can be approximated as a perfectly-insulating membrane--while the interior of the cell is assumed to be conducting; that is, its specific resistance is less than .rho.. A uniform potential gradient E.sub.0 is impressed on the medium containing the cell. Although the cited paper does not give the derivation, it can be shown that the maximum potential difference occurs between the poles of the spherical cell, in the direction of E.sub.0. This potential difference, V.sub.max is EQU V.sub.max =3a.sub.0 E.sub.0
Presumably, this potential would be divided equally between the membranes at either pole. If the membrane has a thickness .tau., then the potential gradient E.sub.p at the poles (where this gradient has its maximum value) would be EQU E.sub.p =(3/2)(a.sub.0 /.tau.)E.sub.0
Thus, the gradient in the membrane increases with an increase in the overall size (represented by the radius a.sub.0) of the cell and decreases with an increase in the membrane thickness.
Below a certain critical gradient--which depends upon the type of cell and is of the order of 10 kV/cm--the porosity induced in the cell membrane is reversible. That is, when the gradient is removed, the membrane regenerates its properties. Whereas, for values of gradient above the critical value, there is an increasing probability that the cell will be destroyed. There is also evidence that--for a given value of potential gradient--the effect increases with increasing time of application.
Various systems for applying high potential gradients to a medium containing microbes are disclosed in U.S. Pat. No. 5,048,404 to Bushnell et al. and in U.S. Pat. No. 5,235,905 to Bushnell et al. Apparatus for inactivation of viruses using pulsed high electric field is disclosed in an article by Mizuno et al. entitled "Inactivation of Viruses using Pulsed High Electric Field" at Conference Record, Annual Meeting, IEEE Industry Applications Society, page 674, 1990.
______________________________________ Technical Bibliography ______________________________________ 1949 H. Burton, National Institute for Research in Dairying, Paper #1041, Reading, England 1961 Doevenspeck, Fleischwirstschaft 13, 986 1967 A. J. H. Sale & W. A. Hamilton, Biochimica & Biophysica Acta 148, 781 1967 W. A. Hamilton & A. J. H. Sale, Biochimica & Biophysica Acta 148, 789 1968 A. J. H. Sale & W. A. Hamilton, Biochimica & Biophysica Acta 163, 37 1971 Roland Benz & K. Janko, Biochimica & Biophysics Acta 455, 721 1973 J. M. Crowley, Biophysics Journal 13, 711 1974 Ulrich Zimmermann, Gunther Pilwat & F. Riemann, Biophysics Journal 14, 881 1974 S. H. White, Biophysics Journal 14, 155 1974 Ulrich Zimmermann, Gunther Pilwat & F. Riemann, Dielectric Breakdown in Cell Membranes, in: Membrane Transport in Plants, p. 146, Springer, Berlin 1975 H. G. L. Coster & Ulrich Zimmermann, Biochimica & Biophysica Acta 382, 410 1975 F. Riemann, Ulrich Zimmermann & Gunther Pilwat, Biochimica & Biophysica Acta 394, 449 1975 Gunther Pilwat, Ulrich Zimmermann & F. Riemann, Biochimica & Biophysica Acta 406, 424 1975 J. Requena & D. A. Haydon, Biophysics Journal 15, 77 1976 Ulrich Zimmermann, Gunther Pilwat, G. Beckers & F. Riemann, Bioelectrochemistry & Bioenergetics 3, 58 1976 Roland Benz & P. Lauger, Journal of Membrane Biology 27, 171 1977 Ulrich Zimmermann, F. Beckers & H. G. L. Coster, Biochimica & Biophysica Acta 464, 399 1978 G. Boheim & Roland Benz, Biochimica & Biophysica Acta 507, 262 1978 J. Vienken, E. Jeltsch & Ulrich Zimmermann, Cytobiology 17, 182 1979 Roland Benz & Ulrich Zimmermann, Journal of Membrane Bilogy, 48, 181 1980 Roland Benz & Ulrich Zimmermann, Biochimica & Biophysica Acta 597, 637 1980 Ulrich Zimmermann, J. Vienken & Gunther Pilwat, Bioelectrochemistry & Bioenergetics 7, 553 1980 H. Hulsheger & Eberhard Neumann, Radiation & Environmental Biophysics 18, 281 1980 Ulrich Zimmermann, Gunther Pilwat, A Pequeux & R. Giles, Journal of Membrane Biology 54, 103 1981 Ulrich Zimmermann, Peter Scheurich, Gunther Pilwat & Roland Benz, Angewandte Chemie 93, 332 1983 H. Hulsheger, J Potel & Eberhard Neumann, Radiation & Environmental Biophysics 20, 53 1986 Akihira Mizuno & Yuji Hori, IEEE Trans. on Indust. Applications 24, 387 1989 Eberhard Niemann, A. E. Sowers & C. A. Jordan, Electroporation and Electrofusion in Cell Biology, Plenum Press, N.Y. 1990 Akihira Mizuno, et al., Conference Record, Ann. Mtg. Industrial Applications Soc., IEEE, p. 713 1991 S. Jayaram, G. S. P. Castle & A. Margaritis, Proc. Annual Mtg. Industry Applications Soc., IEEE, p. 674 1991 Yoichi Matsumoto, Norio Shioji, Tokuki Satake & Akihiro Sakuma, Ibid., p. 652 1991 J. Wilschut & D. Hoekstra, Membrane Fusion, Marcel Dekker N.Y. ______________________________________